Role in Diagnostics
SiDMAP’s technology, which was described at the Molecular Medicine Tri-Conference, is used primarily for mechanism-of-action studies and determining toxicity and tumorigenesis, but it also applies to diagnostics, Dr. Boros says. The technology is being used in humans by administering labeled glucose mixed in water or a fatty acid pill to patients. After an hour or two, a breath test or a finger-prick blood sample is obtained and analyzed to identify biomarkers for a particular disease. A similar method was used to identify Helicobacter pylori bacterium via a specific 13C-labeled substrate, which is associated with more than 90% of peptic ulcers. It resulted in a Nobel Prize for Barry J. Marshall and J. Robin Warren in 2005.
Many of the advances in metabolomics are due to corresponding advances in detection and analysis equipment. Thermo Fisher Scientific (www.thermofisher.com) and Advion Biosciences (www.advion.com), reportedly, are developing more sensitive detection methods for metabolites, and Bio-Rad Laboratories (www.biorad.com) has developed what it considers the holy grail for NMR-based metabolomics.
Thermo Fisher integrates “high-speed, high-performance liquid chromatography; high mass accuracy mass spectrometry; and automated data acquisition and analysis to increase throughput, the sensitivity of detecting metabolic changes in complex samples, and ease of data interpretation,” according to Anne Ferguson, Ph.D., strategic marketing manager. She says the result enables detection of two to 10-fold changes in compound abundance in complex components of body fluids.
The goal, she elaborates, is to “robustly identify subtle differences in metabolic profiles that will promote our understanding of disease and drug safety.”
Thermo Fisher’s Accela™ high-speed chromatography system reduces processing time. “If you consider a small 20-patient study, using normal chromatography, sample processing may take four eight-hour days. The high-speed system may conservatively reduce that time by half,” Dr. Ferguson says, while maintaining high-resolution and retention time reproducibility.
To identify statistically significant differences in the metabolome that correlate to disease, Sieve™ differential expression software was used to compare the chromatographic and spectral information from sets of samples, letting users focus only on the meaningful data, thus yielding more detailed structural analyses.
The LTQ Orbitrap™ hybrid mass spectrometer, another integral component, delivers tandem mass spectrometry and MSn data, offering subfemtomole sensitivity, mass accuracy that is independent of intensity, and many ionization modes. Coupling this with the Mass Frontier™ software provides the ability to “confidently determine the identity and structure of metabolites,” Dr. Ferguson says.
“This is the first time we have used the combination of Sieve software to identify differentially expressed metabolites and Mass Frontier software to determine their structure. We are planning to seamlessly integrate the two software packages,” adds Dr. Ferguson, who detailed steps to taking an integrated approach to metabolomics during a presentation at last month’s International Symposium on Environmental Metabolomics at the University of Louisville in Kentucky.
At Advion Biosciences, Jack Henion, Ph.D., CSO and chairman, is championing an automated nanoelectrospray that can be coupled with most mass spectrometers for metabolomic studies. The TriVersa™ Nanomate, he says, is adept at detecting and identifying chemicals for qualitative and quantitative applications.
The benefits for metabolomic studies include reduced sample consumption, improved detection sensitivity, and enhanced data quality and information for proteomic studies, which lead to gains in unknown compound characterization.
This is particularly important when dealing with complex samples. “There are high levels of albumin in blood, for example, and other important biological compounds that are trillions of times lower,” points out Dr. Henion, who also spoke at the Louisville meeting. Fractionation simplifies detection, identification, and analysis of those less abundant substances by infusing the sample continuously to the mass spectrometer and averaging the signal until sufficient spectra are acquired to accurately measure mass and gain compound structure information. With signal averaging, “noise is averaged to zero, while analyte information increases,” he adds. With a dynamic range that may exceed 10,000:1, automated nanoelectrospray mass spectrometry exhibits “greater than 10 times more sensitivity than LC/MS/MS,” according to Advion.
Tests comparing automated nanoelectrospray with LC/MS showed a fourfold to fivefold throughput increase and, typically, a 100-fold reduction in required analyte quantities, so picograms of compound may be sufficient to achieve accurate measurements.